Effect of cAMP and FSH on the production of estrogens by rat fetal gonads cultured in vitro

The regulation of aromatase activity by cAMP and FSH has been demonstrated in the prepubertal rat testis and ovary, and the question posed whether this regulation was already operative at foetal stages. In the present study, testes and ovaries from 17- to 21-day-old rat foetuses were cultured in vitro in the presence of [3H]-testosterone and in the presence or absence of cAMP or FSH. Oestrone and oestradiol formed from [3H]-testosterone were measured by double isotopic dilution and recrystallization to constant specific activity. Aromatase activity was augmented in both gonads by cAMP, but only in the testis by FSH. Thus, the regulation of aromatase activity by FSH begins earlier in the testis than in the ovary.     

Effect of gonadotropins and prostaglandin on 3' : 5'-cyclic AMP synthesis in young rat testis.

Formation of cAMP has been studied in neonatal rat testis. In the presence of aminoophylline, LH and PGE1 stimulated cAMP levels during the entire postnatal period. The highest stimulation was found in the testis of 2-day-old rats after-which cAMP synthesis diminished with advancing age. FSH had no stimulatory effect on cAMP level in 5-day-old rats.     

Development of fetal rat ovaries responsiveness to LH during organ culture.

The responsiveness of fetal neonatal rat ovaries to LH was investigated in vitro using three complementary approaches. First, steroid production was assessed after culture. In control media, detectable levels of estrogens (estradiol and estrone) and progesterone were only observed from day 6 postpartum and during the second week of life respectively. In the presence of LH (100 ng/ml) ovaries produced both estrogens and progesterone from day 4 postpartum and the response to LH was enhanced with IBMX supplementation in the medium. Second, 3 beta-HSD activity was measured with either LH or (Bu)2 cAMP (1mM). Irrespective to the time-period studies (Bu)2 cAMP stimulated this enzyme whereas the stimulation with LH occurred only from day 5 postpartum Third, specific hCG binding was assessed and we found that it occurred only on days 7 and 10. However, when fetal ovaries were pretreated for 48 h with (Bu)2 cAMP, a specific hCG binding could be detected and progesterone production was enhanced in response to LH. An effect of the nucleotide via a stimulation of the neuraminidase activity did not seem to be involved. Lastly treatment of 18-day-old fetal ovaries with cholera toxin (10nM) or forskolin (1 microM) was found to stimulate progesterone production and VIP (0.1 to 1 microM) stimulated both the 3-HSD activity and the estradiol production. These data suggest that the absence of steroidogenic response to LH before day 4 postpartum could be explained by the absence of receptors, though the LH transmembrane signal-transduction system is functional in fetal ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of follicle-stimulating hormone and luteinizing hormone in controlling estradiol synthesis by the testis of the infant rat

Experiments were conducted to assess the relative contribution of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to the regulation of estradiol secretion by the testis of the 12-day-old rat. In an in vivo model system, stimulation of the whole testis with NIH-FSH-S13 (5% LH activity) caused an 8-fold increase in testosterone secretion within 1 h followed by a 5-fold increase in estradiol secretion. Qualitatively, similar findings were obtained from whole testes incubated in tissue culture medium 199. The in vitro system was used to further examine the response of the testes to LH and FSH. Testes exposed to a variety of doses of LH or 10 ng/ml of highly purified FSH (3 X 13, 1% LH activity) showed no change in estradiol secretion. However, a synergistic effect was observed when purified FSH and LH were combined, provided the LH concentration exceeded 25 pg/ml. It is suggested that FSH secretion in infant rats maintains the aromatizing capacity of the seminiferous tubule at a level such that availability of aromatizable substrate becomes a major factor in the rate of tubular estrogen formation.     

Cultivation of rat granulosa cells in a serum-free chemically defined medium--a useful model to study lipoprotein metabolism

We have developed a chemically defined, serum-free medium for the culture of rat granulosa cells. This medium contains Dulbecco's modified Eagle's medium/Ham's nutrient F12 (DME:F12) (1:1) plus insulin (2 micrograms/ml), hydrocortisone (100 ng/ml), transferrin (5 micrograms/ml) and fibronectin (2 micrograms/cm2). Granulosa cells grown in this medium have an absolute requirement for added cholesterol-rich lipoproteins for steroidogenesis. When cells are cultured in basal medium, progestin production is low; when cells are cultured in the presence of follicle-stimulating hormone (FSH) or dibutyryl cAMP [Bu)2 cAMP), progestin secretion is increased 10-100-fold. Both heterologous and homologous lipoproteins synergistically increased the effects of (Bu)2 cAMP or FSH: e.g., addition to the medium of human (h)-HDL3 produced a significant increase in both basal (approx. 15-fold) and (Bu)2 cAMP-stimulated (approx. 1000-2000-fold) progestin production. LDL were less effective than HDL at equivalent concentrations of lipoprotein cholesterol. FSH invoked changes similar to that of (Bu)2 cAMP, although the magnitude of the FSH-induced change was less dramatic than that seen with (Bu)2 cAMP. The effect of h-HDL3 and h-LDL on both basal and hormone-stimulated progestin production was concentration- and time-dependent. The maximum effect of h-HDL3 was achieved at a protein concentration of 500 micrograms/ml, with an ED50 of approx. 90 micrograms/ml. In contrast, h-LDL was most effective at a concentration of 30-40 micrograms protein/ml. Likewise, rat (r-)HDL and r-LDL supported steroidogenesis in a concentration-dependent manner. Maximal responses to all additions were observed after 72 h of treatment. Granulosa cells secreted 20 alpha-hydroxypregn-4-ene-3-one as the predominant steroid in response to (Bu)2 cAMP. However, with the addition of h-HDL3, the major secreted product was progesterone. In conclusion, rat granulosa cells maintained in the described serum-free medium are exquisitely sensitive to supplied cholesterol-rich lipoproteins. When cultured in the presence of both lipoproteins and stimulatory agents, they produce from 1000-2000-times the progestins made by comparable cells maintained in medium alone. This responsiveness of the cells to both lipoprotein and hormone stimulation makes them uniquely suitable for studies involving the uptake and metabolism of lipoproteins during steroidogenesis.     

Estrogen production by fetal rat gonads

Aromatase activity in fetal rat testes and ovaries was demonstrated by the conversion of tritiated testosterone or 19-hydroxyandrostenedione into estrone and estradiol, which were identified and quantified by double isotopic dilution and recrystallization to constant specific activity. Testes formed mostly estradiol, ovaries mostly estrone. Aromatase activity was stimulated by cAMP in both the testes and ovaries as early as 17 days of fetal life. Stimulation by FSH was noted at this same stage in the testis, but not before 3–4 days after birth in the ovary. LH was without effect on aromatase activity in both kinds of gonads. Basal estrogen secretion was non-existent or undetectable in both the testes and ovaries in fetal stages. In the presence of cAMP and as early as 17 days of fetal life, the testes released estradiol, as early as 14 days the ovaries released estrone. Estrogen secretion was stimulated by LH and FSH at fetal stages in the testis and at infantile stages in the ovary. Responsiveness to gonadotrophins closely followed the appearance of the receptors.     

Influence of A23187 and dibutyryl cyclic AMP on progestagen production by rat granulosa cells in vitro

The gonadotrophic regulation of progesterone production by rat granulosa cells was examined in a chemically-defined medium containing FSH, dibutyryl cyclic AMP [Bu)2cAMP) and the calcium ionophore, A23187. FSH and A23187 alone significantly enhanced the production of pregnenolone, progesterone and its metabolite, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) from endogenous substrate(s). Stimulation of progesterone production by A23187 was accompanied by an increase in 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) but not 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity, as attested by enhancement of the metabolism of exogenous pregnenolone to progesterone but not of progesterone to 20 alpha-OH-P. In contrast, although (Bu)2cAMP increased pregnenolone and progesterone production and the metabolism of exogenous progesterone to 20 alpha-OH-P, it failed to stimulate the conversion of exogenous pregnenolone to progesterone. The increase in progesterone production and in the conversion of exogenous pregnenolone to progesterone by FSH and A23187 was concentration- and time-dependent. Whereas maximal stimulation of de-novo progesterone synthesis by FSH was evident by 6 h (earliest time examined), a significant increase in the conversion of exogenous pregnenolone to progesterone in the presence of FSH or the ionophore was not noted until 12 h of incubation. Although a small but significant increase in progesterone production was also noted as early as 6 h of incubation in the presence of the calcium ionophore, this was markedly smaller than that elicited by FSH. We conclude that the calcium ionophore A23187 and (Bu)2cAMP have similar as well as distinct effects on progesterone production in rat granulosa cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphasic response to 3',5'-cyclic adenosine monophosphate (cAMP) at the messenger ribonucleic acid level for a regulatory subunit of cAMP-dependent protein kinase

In the present study we have examined the effect of long-term stimulation with (Bu)2cAMP on mRNA levels for the hormone responsive regulatory subunit (RII beta) of cAMP-dependent protein kinase in cultured rat Sertoli cells. The effects of the same treatment on two other mRNAs [androgen binding protein (ABP) and cellular retinol binding protein (cRBP)], shown to be regulated by cAMP, were examined simultaneously. The addition of (Bu)2cAMP (0.1 mM) to primary Sertoli cell cultures, for 14 and 24 h, caused a 50- to 60-fold stimulation in the steady state levels of mRNA for RII beta. During the same period of stimulation, we also observed a significant increase (2- to 3-fold) in the mRNA levels for ABP, and a 80% decrease in the mRNA levels for cRBP. Continued stimulation for 36 and 48 h was associated with a significant time-dependent decrease in the mRNA level for RII beta, in spite of the continuous presence of (Bu)2cAMP (0.1 mM) in the medium. This reduced response by long term stimulation with (Bu)2cAMP appears to be specific for RII beta, since mRNA for ABP remained elevated and mRNA for cRBP remained depressed during the entire period of cAMP stimulation. Our data demonstrate the presence of a biphasic type of regulation at the mRNA level, specific for the regulatory subunit RII beta of cAMP-dependent protein kinase. This response may be analogous to the desensitization mechanisms observed at other levels of the cAMP signalling pathway. For proteins constituting part of the signal transduction pathway this type of biphasic regulation, may be particularly important in maintaining homeostasis in the cell.     

Prostaglandin F2 alpha inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F2 alpha (PGF2 alpha). The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF2 alpha. A dose-dependent inhibition by PGF2 alpha (0.5-50 microM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165-165 microM). The stimulation by epinephrine on progesterone production was inhibited by PGF2 alpha (5 microM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF2 alpha can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF2 alpha shows in this respect the same age dependent inhibitory pattern as in relation to LH stimulation.     

Ontogeny of pituitary gonadotrophin secretion in the fetal and post-natal pig in response to LHRH in vitro

Monolayer cultures of anterior pituitary cells from male or female pigs of 60, 80, 105 days of fetal life or of 60, 160 and 250 days of post-natal life were prepared and treated with LHRH (1 pM to 10 nM). Dose-related increases of LH were first seen at 80 days of gestation in both sexes, while only female fetuses responded to maximal LHRH at 60 days. Basal and stimulated LH release doubled in cultures from 105-day-old fetuses when compared with those at 80 days. Compared to late fetal stages LH release was 20- to 30-fold higher in cell cultures from 60-day-old (post-natal) donors without further change during the post-natal period. In all pre- and post-natal age groups basal and maximal LH release of pituitary cells from males was lower than that of females. FSH stimulation started in male and female cells at 80 days of gestation only at LHRH concentrations exceeding or equal to 0.1 nM. By 105 days FSH secretion was dose-related and pituitary cells of females responded with higher FSH values than did those of males. In general, post-natal cells released much higher amounts of FSH than did prenatal cells. Basal and maximal release of FSH decreased during post-natal development in both sexes. Basal as well as maximal FSH release of cultures from female donors was higher than that found in cultures from male donors. Determination of total LH and FSH content in fetal pituitary cell cultures indicated that the developmental increase in gonadotrophin release potential is a function of the total gonadotrophin content in vitro. We conclude that (1) the in-vitro release of gonadotrophins to LHRH is dose-, age- and sex-dependent; (2) in the female fetal pig LH responsiveness develops earlier than FSH responsiveness; (3) apparently, these maturational changes mainly reflect alterations in pituitary gonadotrophin content; and (4) there is no simple relationship between in-vitro release and circulating gonadotrophins.     

Action of Mn2+ and vanadium compounds on hormone and forskolin induced stimulation of juvenile rat ovarian adenylate cyclase

The effect of forskolin on the hormonal (LH, FSH) activation and on the stimulation provided by other effectors (Gpp(NH)p,NaF) of the juvenile rat ovarian adenylate cyclase was investigated. Forskolin exhibited a synergistic action with LH, FSH and Gpp(NH)p but not with NaF. Addition of Ca2+ was inhibitory over a concentration range from 10(-5) to 10(-2) M whereas EGTA enhanced the response at 5.10(-5) M and inhibited it at higher concentration. The cAMP production was increased by addition of Mn2+ at low concentration (up to 5 mM) but markedly decreased at higher concentration (30 mM). FSH induced cAMP production was completely abolished at 30 mM Mn2+. The effect of vanadyl ion was very similar to that of Mn2+ Vanadate anion on the contrary was without effect on FSH stimulation.     

Effect of prostaglandins on ornithine decarboxylase activity in the testis of immature rat

Intratesticular injection of prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rats. PGE2 at a dose of 10 microgram per testis was maximally effective 2 hours after the injection. Dibutyryl cyclic AMP (cAMP) and 1 methyl, 3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor, also stimulated ODC activity. Simultaneous injection of PGE2 and FSH or LH caused additional stimulation of ODC activity. Similarly injection of PGE2 in addition to cAMP or MIX also caused increased stimulation of ODC. Indomethacin (IM, 60 microgram/testis) inhibited LH, FSH or cAMP induced ODC activity. However, IM at the same dose inhibited the synthesis of total proteins. These results suggest that PGE2 and PGF2 alpha stimulate the activity of ODC. The action of prostaglandins may be independent of the action of gonadotropic hormones. cAMP appears to mediate the action of prostaglandins in the testis of rat.     

Effects of polyamines on the release of gonadotropin-releasing hormone and gonadotropins in developing female rats

Polyamines, putrescine (PUT), spermidine (SPD), spermine (SPM), and agmatine (AGM), are polycationic amines related to multiple cell functions found in high concentrations during the development of hypothalamus and pituitary. In previous works, we demonstrated that alpha-difluoromethylornithine (DFMO), an inhibitor of polyamines biosynthesis, induced a delay in puberty of female rats, accompanied by high, sustained follicle-stimulating hormone (FSH) levels during the infantile period. Also, DFMO treatment induced changes in polyamine concentration both in hypothalamus and pituitary of rats, mainly a decrease of PUT and SPD, an increase in SPM, and no change in AGM. In the present work, we investigated the direct effects of polyamines on the secretion of hypothalamic GnRH and pituitary gonadotropins in 6- and 15-day-old female rats. In 6-day-old animals, in vitro incubations with PUT, SPD, and AGM of hypothalami or anterior pituitaries were able to inhibit GnRH, FSH, and leutinizing hormone (LH) secretion, respectively. SPM showed a nonspecific transient inhibitory effect on FSH. When challenged with either high K(+) (hypothami) or GnRH (pituitaries), the tissues incubated in the presence of polyamines showed no differences when compared with their controls. No effects of polyamines in 15-day-old rats in either tissue were observed. Pituitary cell cultures of 6-day-old animals incubated with DFMO for 4 days showed a significant increase in FSH, but not in LH. We conclude that high PUT, SPD, and AGM levels during the first 10 days of life are important for the development of the hypothalamic-hypophyseal unit, probably related to an inhibitory effect on GnRH and gonadotropins. Therefore, polyamine participation, especially PUT and SPD, is of importance in the regulation of GnRH and gonadotropin secretion in the neonatal and infantile periods, critical stages in the establishment of sexual differentiation.     

Autocrine role of estrogens in the augmentation of luteinizing hormone receptor formation in cultured rat granulosa cells

The effects of estrogens on gonadotropin-stimulated luteinizing hormone (LH) receptor formation were examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of follicle-stimulating hormone (FSH) in the presence or absence of native and synthetic estrogens. Follicle-stimulating hormone stimulated LH receptor formation in a dose-dependent fashion, and estrogens enhanced the FSH-stimulated LH receptor content by decreasing the apparent ED50 of FSH. At 6.25 ng/ml FSH, the enhancement in LH receptor was estrogen dose dependent, with an ED50 value of about 3 X 10(-9) M for 17 beta-estradiol. The increased LH receptor content seen in cells treated with FSH and estrogen was correlated with increased cAMP production by these cells in response to LH stimulation. Time course studies revealed enhancement of FSH-stimulated LH receptor induction at 48 and 72 h of culture. Granulosa cells were also cultured with FSH for 2 days to induce functional LH receptors, then further cultured for 3 days with LH in the presence or absence of estrogens. At 30 ng/ml LH, increasing concentrations of estrogens maintained LH receptor content in a dose-dependent fashion, with their relative estrogenic potencies in keeping with reported binding affinities to estrogen receptors. An autocrine role of estrogens on LH receptor formation was further tested in granulosa cells treated with FSH and an aromatase substrate (androstenedione) to increase estrogen biosynthesis. Cotreatment with semipurified estrogen antibodies partially blocked the FSH stimulation of LH receptors, whereas nonimmune serum was ineffective. Also, inclusion of diethylstilbestrol prevented the inhibitory effect of the estrogen antibodies. Thus, local estrogens in ovarian follicles may play an autocrine role in granulosa cells to enhance LH receptor formation and to increase granulosa cell responsiveness to the LH surge, with subsequent ovulation and adequate corpus luteum formation.     

Regulation of mRNA expression of 3 beta-hydroxy-5-ene steroid dehydrogenase in porcine granulosa cells in culture: a role for the protein kinase-C pathway

We studied the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase-C, on porcine granulosa cells in culture. PMA as well as cholera toxin, forskolin, and hCG increased cAMP accumulation. PMA further augmented the elevation in cAMP accumulation induced by cholera toxin, forskolin, and hCG. In the same cell culture model, hCG induced a time-dependent increase in the 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) mRNA levels with a maximal 3-fold stimulation obtained at 8-16 h of incubation with 1 IU hCG/ml. PMA inhibited the increase in 3 beta HSD mRNA levels induced by hCG in a dose-dependent manner. The phorbol ester also inhibited the increase in 3 beta HSD mRNA levels stimulated by LH as well as cholera toxin and forskolin and the cAMP analogs (Bu)2cAMP and 8-bromo-cAMP. Activation of protein kinase-C by mezerein similarly inhibited hCG stimulation of 3 beta HSD mRNA levels. The present data indicate that activation of the protein kinase-C pathway induces generation of cAMP, but causes a near-complete inhibition of the stimulatory effects of hCG, LH, forskolin, cholera toxin, and cAMP analogs on 3 beta HSD mRNA levels in porcine granulosa cells in culture.     

Gonadotropic regulation of prostaglandin production by ovarian follicular cells of the pig

Prepubertal gilts were treated with 750 IU pregnant mare's serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa cells (GC) and theca interna cells (TC) from follicles of gilts 72 h (GC-72 and TC-72, respectively) and 108 h (GC-108 and TC-108 h, respectively) after PMSG treatment were cultured for 0, 12, 24, and 36 h in medium with or without luteinizing hormone (LH), dibutyryl cyclic adenosine 3',5'-monophosphate [Bu)2cAMP), calcium ionophore (A23187), and/or arachidonic acid (AA), and the production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. TC-72 was the principal source of PGs 72 h after PMSG. At 108 h, the production of PGE and PGF by GC was increased 10- and 30-fold, respectively, whereas corresponding increases by TC were 2-fold. LH and A23187 significantly stimulated PGE and PGF production by both GC-72 and TC-72, but only thecal PG production was stimulated by (Bu)2cAMP. LH had minimal or no effect on PG production by GC-108 and TC-108, but A23187 (GC-108, TC-108) and (Bu)2cAMP (TC-108) were stimulatory. Basal PG production by GC-72, GC-108, and TC-108 was stimulated by AA. However, production by GC and TC cultured in medium containing AA and LH, A23187, or (Bu)2cAMP was not different from that produced by AA alone. These findings suggested that GC and TC can synthesize PGs in vitro, but AA availability is rate-limiting in GC. After exposure to hCG in vivo, the capacity of both cell types to produce PGs is increased but is limited by AA availability.(ABSTRACT TRUNCATED AT 250 WORDS)